Microbial Profiling Using Mothur Pipeline

Snakemake workflow


Snakemake workflow



Mothur Reference databases

  1. Mothur-based SILVA reference files[1].
  2. Mothur-based RDP reference files[2]. Note: The RDP database is to classify 16S rRNA gene sequences to the genus level.
  3. ZymoBIOMICS Microbial Community Standard (Cat # D6306)[3]. The ZymoBIOMICS Microbial Community DNA Standard is designed to assess bias, errors and other artifacts after the step of nucleic acid purification.


Mothur classification methods

There are four methods that can be used to profile microbial communities present in a sample. Here we briefly decribe each method:

1.Classify OTUs

  • OTUs (Operational Taxonomic Units (OTUs)) are clusters of similar sequences and are commonly accepted as analytical units in microbial profiling when using 16S rRNA gene markers.

2. Classify Phylotypes

  • A phylotype in microbiome research is a DNA sequence or group of sequences sharing more than an arbitrarily chosen level of similarity of a 16S rRNA gene marker.

3. Classify ASVs

  • ASVs Amplicon Sequence Variants (ASVs)in microbiome research is any inferred single DNA sequences recovered from a bioinformatics analysis of 16S rRNA marker genes.
  • ASV is typically really a cluster of sequences that are one or two bases apart from each other.

4. Classify Phylogenies

  • Microbial phylogenies are from gene sequence homologies. Models of mutation determine the most-likely evolutionary histories.


Preliminary OTU analysis using Mothur

The preliminary analysis (alpha_beta_diversity rule) is part of the bioinformatics analysis. It includes:

  • Creating reads count for each group.
  • Subsampling for downstream analysis.
  • Rarefaction.
  • Computing Alpha diversity metrics.
  • Computing Beta diversity metrics.
  • Getting sample distances.
  • Constructing sample phylip tree.
  • Generating ordination matrices including PCoA and NMDS.

…IN PROGRESS…





References

[1]
Mothur-based silva reference files. Retrieved from https://mothur.org/wiki/silva_reference_files/
[2]
Mothur-based RDP reference files. Retrieved from https://mothur.org/wiki/rdp_reference_files/
[3]
ZymoBIOMICS microbial community DNA standard (cat # D6306). Retrieved from https://www.zymoresearch.com/zymobiomics-community-standard
[4]
Buza, T. M., Tonui, T., Stomeo, F., Tiambo, C., Katani, R., Schilling, M., … Kapur, V. (2019). iMAP: An integrated bioinformatics and visualization pipeline for microbiome data analysis. BMC Bioinformatics, 20. https://doi.org/10.1186/S12859-019-2965-4


Appendix

Project main tree

.
├── LICENSE.md
├── README.md
├── REFER
│   ├── dags
│   ├── data
│   ├── envs
│   ├── images
│   ├── imap-bioinformatics.Rproj
│   ├── index.html
│   ├── render.R
│   ├── report.html
│   ├── resources
│   ├── results
│   ├── rules_dag.sh
│   ├── smk_html_report.sh
│   └── workflow
├── config
│   ├── config.yml
│   ├── pbs-torque
│   ├── samples.tsv
│   ├── slurm
│   └── units.tsv
├── dags
│   ├── rulegraph.png
│   └── rulegraph.svg
├── data
│   ├── README.md
│   ├── metadata
│   ├── mothur
│   ├── reads
│   ├── references
│   └── test
├── images
│   ├── bioinformatics.png
│   ├── bkgd.png
│   └── smkreport
├── index.Rmd
├── library
│   ├── apa.csl
│   ├── export.bib
│   ├── imap.bib
│   └── references.bib
├── report.html
├── results
│   └── project_tree.txt
├── styles.css
├── tree.sh
└── workflow
    ├── Snakefile
    ├── envs
    ├── rules
    └── scripts

26 directories, 26 files

Screenshot of interactive snakemake report

The interactive snakemake HTML report can be viewed by opening the report.html using any compatible browser. You will be able to explore the workflow and the associated statistics. You can close the left bar to get a more expansive display view.

Troubleshooting of FAQs

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